We are studying nervous system specific kinases and phosphatases to understand the role of neurofilament phosphorylation. Previously, we have shown that second messenger dependent and independent kinases can phosphorylate the neurofilament proteins (NFPs) in vitro; however, the kinases involved in phosphorylation in vivo are unknown. In order to determine the kinases involved in this phosphorylation, we have analyzed the endogenous phosphorylation sites in carboxy-terminal tail domain of high molecular weight neurofilament protein (NF-H), where most of the in vivo phosphorylation takes place. These studies showed that serines (s) in all three KSP motifs: KSPXK, KSPXXK and KSPXXXK are phosphorylated. Cyclin-dependent kinase, Cdk-5, from rat spinal cord or bovine brain, specifically phosphorylated KSPXK motifs. Purification of this kinase from rat spinal cord showed that it is associated with a protein of 67KDa (p67). Removal of this protein from the complex resulted in a considerable loss of the kinase activity which could be restored by adding back the purified or bacterially expressed p67. The complete amino acid sequence of p67 deduced from a number of cDNA clones from rat brain cDNA libraries appeared to be identical to Munc-18, a synaptic vesicle complex protein. The p67 transcript expression begins early in development and is restricted to the nervous tissue. Immunohistochemical staining with an amino-terminal peptide-specific antibody indicated that p67 is expressed exclusively in neurons. Localization in brain tissue and in cultured rat hippocampal neurons demonstrated that p67 is highly enriched in axons. We also investigated the expression of cdk-5, p67 and phosphorylated NF-H during development of the rat cerebellum from postnatal day 2 to adult using specific antibodies to these molecules. We could demonstrate that at all stages the epitopes for Cdk-5 and phosphorylated NF-H were colocalized in developing afferent fibers (mossy and climbing fibers) as well as basket cell fibers surrounding the Purkinje cells. Purkinje cell bodies, on the other hand, contained high levels of dephosphorylated NF-H and NF-M. These results are consistent with the hypothesis that cdk5 regulates the phosphorylation of some of the KSP motifs in neurofilament proteins.